Sulfur-containing compounds such as cysteine, homocysteine, methionine or S-adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals. In particular methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Nevertheless most of the methionine which is produced is added to animal feed.
With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Chemically D,L-methionine is commonly produced from acrolein, methyl mercaptan and hydrogen cyanide. Nevertheless the racemic mixture does not perform as well as pure L-methionine, as for example in chicken feed additives (Saunderson, C. L., (1985) British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine e.g. through the acylase treatment of N-acetyl-D,L-methionine which increases production costs dramatically. The increasing demand for pure L-methionine coupled to environmental concerns render microbial production of methionine attractive.
Microorganisms have developed highly complex regulatory mechanisms that fine-tune the biosynthesis of cell components thus permitting maximum growth rates. Consequently only the required amounts of metabolites, such as amino acids, are synthesized and can usually not be detected in the culture supernatant of wild-type strains. Bacteria control amino acid biosynthesis mainly by feedback inhibition of enzymes, and repression or activation of gene transcription. Effectors for these regulatory pathways are in most cases the end products of the relevant pathways. Consequently, strategies for overproducing amino acids in microorganisms require the deregulation of these control mechanisms.
The pathway for L-methionine synthesis is well known in many microorganisms. Methionine is derived from the amino acid aspartate, but its synthesis requires the convergence of two additional pathways, cysteine biosynthesis and C1 metabolism (N-methyltetrahydrofolate). Aspartate is converted into homoserine by a sequence of three reactions. Homoserine can subsequently enter the threonine/isoleucine or methionine biosynthetic pathway. In E. coli entry into the methionine pathway requires the acylation of homoserine to succinyl-homoserine. This activation step allows subsequent condensation with cysteine, leading to the thioether-containing cystathionine, which is hydrolyzed to give homocysteine. The final methyl transfer leading to methionine is carried out by either a B12-dependent or a B12-independent methyltransferase. Methionine biosynthesis in E. coli is regulated by repression and activation of methionine biosynthetic genes via the MetJ and MetR proteins, respectively (reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds), 1996, Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology; Weissbach et al., 1991 Mol. Microbiol., 5, 1593-1597). MetJ together with its corepressor S-adenosylmethionine is known to regulate the genes metA, metB, metC, metE and metF. Other genes encoding enzymes implicated in methionine production, such as glyA, metE, metH and metF are activated by MetR whereas metA is repressed by MetR. The corresponding enzymes are all involved in the production and the transfer of C1 units from serine to methionine. GlyA encoding serine hydroxymethyltransferase catalyzes the conversion of serine to glycine and the concomitant transfer of a C1 unit on the coenzyme tetrahydrofolate (THF). The C1 unit in form of methylene-THF needs to be reduced to methyl-THF before it can be transferred on homocysteine to yield methionine. This reaction is catalyzed by the MetF protein. Transfer of the methylgroup is either catalyzed by MetH via vitamin B12 or directly by MetE. The MetH enzyme is known to have a catalytic rate that is hundred times higher than the MetE enzyme. In the absence of vitamin B12 and thus active MetH, MetE can compose up to 5% of the total cellular protein. The presence of active MetH reduces MetE activity probably by reducing the amount of homocysteine that normally activates the transcription of metE via MetR. Therefore the production of methionine via MetH saves important resources for the cell by not expressing large quantities of MetE. An accumulation of homocysteine is toxic for E. coli (Tuite et al., 2005 J. Bacteriol, 187, 13, 4362-4371) and at the same time has a negative, regulatory effect on metA expression via MetR. Thus a strong expression of the enzymes MetH and/or MetE is clearly required for efficient methionine production.
In E. coli reduced sulfur is integrated into cysteine and then transferred onto the methionine precursor O-succinyl-homoserine, a process called transulfuration (reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology). Cysteine is produced from O-acetylserine and H2S by sulfhydrylation. The process is negatively feed-back regulated by the product, cysteine, acting on serine transacetylase, encoded by CysE. N-acetyl-serine, which is spontaneously produced from O-acetyl-serine, together with the transcription factor CysB activates genes encoding enzymes involved in the transport of sulfur compounds, their reduction to H2S and their integration in the organo-sulfur compound cysteine, which as methionine is an essential amino acid.
In the absence of cysteine, MetB catalyzes the conversion of the methionine-precursor O-succinyl homoserine into ammonia, α-ketobutyrate and succinate, a reaction called γ-elimination (Aitken & Kirsch, 2005, Arch Biochem Biophys 433, 166-75). α-ketobutyrate can subsequently be converted into isoleucine. This side reaction is not desirable for the industrial production of methionine, since the two amino acids are difficult to separate. Thus low γ-elimination activity is an important aspect for the industrial production of methionine. The provisional patent application U.S. 60/650,124 filed on Feb. 7, 2005 describes how γ-elimination can be reduced by optimizing the enzyme MetB. Optimizing the flow of cysteine biosynthesis can also reduce γ-elimination and thus the production of the byproduct isoleucine and constitutes an embodiment of this invention.